Gene-based delivery systems as vaccines are highly promising for infectious diseases and cancer. Unique advantages of recombinant adeno-associated virus vector (rAAV) in the development of effective cancer vaccines include induction of sustained, high-level transgene expression, absence of viral structural proteins, non-pathogenicity, and absence of toxicity. The use of alternate AAV serotype capsids to overcome target cell refractoriness and molecular modifications in the rAAV genome to effect early onset of transgene expression further increases the utility of this vector. Although gene delivery systems have proven efficacious as vaccines in the treatment of cancer, the major limitations for effective immune activation against tumors have been diminution of antigen expression, which curtails robust effector activation, and vector-specific immune responses, which eliminate transgene-expressing cells prior to the induction of strong anti-tumor immunity. By studies outlined in this application, we propose to exploit the advantages of rAAV as a tumor vaccine using a highly relevant preclinical mouse model of melanoma. Studies so far in our lab with rAAV encoding a prototypic tumor antigen and adapting several modifications including the use of serotype 6 capsid, rAAV containing double-stranded genome, multiple transductions with rAAV6 followed by dendritic cell activation in cultures, and the use of synthetic oligonucleotide and plasmid DNA adjuvant following direct rAAV immunizations have resulted in significant antigen-specific, Th1-associated immune responses and anti-tumor activity in C57BL6/J mice. More interestingly this approach was successful in breaking immune tolerance to a self tumor antigen. The current proposal will focus on developing this vector paradigm to more rigorously test the induction of both protective and therapeutic tumor immunity in a clinically relevant mouse melanoma model using tyrosinase-related protein (TRP) 2 antigen by ex vivo and direct in vivo vaccine strategies. Mechanisms of tumor immunity will be examined in vivo. Further, the influence of pre-existing anti-viral immunity on the development of effective anti-tumor immunity will be studied. Public Health Relevance: Results of this translational study are anticipated to rapidly enable the initiation of phase I clinical trial employing rAAV-TRP2 vaccine for melanoma and can form the basis for a novel immunotherapy strategy for other human cancers characterized by overexpression of tumor antigens. Based on the unique advantages of recombinant AAV including sustained high-level expression, non- pathogenicity and low vector immunity, the proposed translational study will develop and determine the potential of rAAV vaccine in combination with oligonucleotide and plasmid DNA immune adjuvant for strong CTL cell response in a mouse melanoma model.